Neuroscience
A subscription to JoVE is required to view this content.
You will only be able to see the first 2 minutes.
The JoVE video player is compatible with HTML5 and Adobe Flash. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. We recommend downloading the newest version of Flash here, but we support all versions 10 and above.
If that doesn't help, please let us know.
Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice
Chapters
Summary January 21st, 2022
Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.
Transcript
This protocol provides step-by-step guidelines for a reproducible route of access to regions of interest in the caudal brainstem and the upper cervical cord. This technique increases precision when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. This technique has been and can be applied to other animal models.
Dr.Veronique VanderHorst, an associate professor of neurology and principal investigator of the VanderHorst Laboratory, will demonstrate this procedure. Make sure that the oxygen flow is directed to the nose cone. Move the mouse to the stereotaxic frame and place the nose in a flexible nose cone.
Place the mouse in the stereotaxic frame using ear bars only. Place lubricant on the eyes. Anteroflex the mouse's head to a 90 degree angle by manually guiding the nose.
To secure this position, place a plastic barrier between the ear bar pillars of the mouse adapter parallel to the pillars with the flat part of the skull serving as the reference. Place the heating pad underneath the mouse and make sure that the neck and the rest of the body are positioned parallel to the table by elevating the body with a small box. Place a drape underneath the body.
Inject a single dose of four milligrams per kilogram Meloxicam slow release subcutaneously at a volume of two microliters per gram of body weight. Clean the surgical incision site first with a 70%alcohol prep pad, then with a Betaine prep pad, and then again with an alcohol prep pad and let it dry. Disinfect hands and put on sterile gloves, then place a drape at the surgical site.
Ensure that the mouse is appropriately anesthetized by pinching the toes or checking the corneal reflex. Reduce isoflurane to maintain the levels at 2.0. Make a one to 1.2 centimeter incision with a surgical 10 blade from the edge of the occipital bone toward the shoulders in one smooth movement.
Carefully make an incision in the midline raphe of the trapezius muscle, exposing the paired longus capitis muscles. Place both retractor hooks between the paired longus capitis muscles, one oriented to the left and the other to the right. Use the blunt laminectomy forceps to separate the left and right bellies of the paired longus capitis muscle, starting from the occiput where the midline is readily visible.
Guide the blunt forceps across the bone of the occiput in the midline down to where it meets the cisternal dura mater and then continue across the dura mater to the atlas. Reposition the retractors and adjust the tension by repositioning the hemostats. Use the blunt laminectomy forceps to separate the muscles further in the midline to obtain a good view of the brainstem and cerebellum.
Repeat the above procedure as needed until the cerebellum and brainstem appear below the dura. Using blunt laminectomy forceps, clear the dura of the small strands of connective tissue by moving the forceps from the midline in a lateral direction until there is a clear view of the brainstem, creating more lateral space. View the dorsal surface of the brainstem with detailed landmarks through the open dura.
Use the angled forceps to grab the dura extending from the occipital bone to the atlas, then use the spring scissors to make a small opening of approximately 0.5 to 1.5 millimeters in the dura. Once the dura is opened, drain excess cerebrospinal fluid with a sterile Q-tip. The obex, the point where the central canal opens into the fourth ventricle, is the standard anterior-posterior and mediolateral zero point.
Position the pipette or syringe to the target using the stereotaxic arm. Lower the arm of the dorsal onto the dorsal surface which forms from the dorsoventral zero point. Then lower the pipette onto the brainstem and inject the solution.
Leave the needle in place for one to five minutes after injection to avoid a needle track when using volumes between three to 50 nanoliters. Then lift the pipette or syringe using the stereotaxic arm and repeat this for multiple targets. Remove the hooks carefully from the surgical field.
The paired longus capitis muscles will fall back into a neutral position fully covering the cisterna magna. Do not close the trapezius muscle and dura mater in the midline as they are too fragile to hold sutures. Close the skin with 3 nylon or polypropylene sutures.
The cisterna magna approach makes it possible to target the caudal brainstem and upper cervical cord structures that are otherwise hard to reach via standard stereotaxic approaches or are prone to inconsistent targeting. In mice, structures such as the hypoglossal nucleus, ventral respiratory group and adjacent reticular formation in the caudal brainstem have been routinely targeted using the cisterna magna approach as illustrated here for the hypoglossal nucleus and the ventromedial medulla. In order to determine the accuracy of the cisterna magna approach versus the standard approach, the distance between the intended and actual target sites in the anteroposterior, mediolateral, and dorsoventral planes for ventral and dorsal regions was measured.
The results show significantly smaller errors in the anteroposterior, mediolateral, and dorsoventral planes compared to the standard approach, highlighting the enhanced accuracy of the cisterna magna approach for these targets. When attempting this protocol, it is critical to make sure that the anteroflexion of the head and the elevation of the body is performed as described. Next, it is important to recognize key landmarks before manipulating the muscle or the dura mater.
If these landmarks are not recognized or if they're lost, it will be challenging to stay oriented and execute the procedure as intended. This technique helped address conceptual questions related to the functional anatomical organization within the caudal brainstem and upper cervical cord.
Related Videos
You might already have access to this content!
Please enter your Institution or Company email below to check.
has access to
Please create a free JoVE account to get access
Login to access JoVE
Please login to your JoVE account to get access
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Please enter your email address so we may send you a link to reset your password.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Your JoVE Unlimited Free Trial
Fill the form to request your free trial.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Thank You!
A JoVE representative will be in touch with you shortly.
Thank You!
You have already requested a trial and a JoVE representative will be in touch with you shortly. If you need immediate assistance, please email us at subscriptions@jove.com.
Thank You!
Please enjoy a free 2-hour trial. In order to begin, please login.
Thank You!
You have unlocked a 2-hour free trial now. All JoVE videos and articles can be accessed for free.
To get started, a verification email has been sent to email@institution.com. Please follow the link in the email to activate your free trial account. If you do not see the message in your inbox, please check your "Spam" folder.